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MicroRNAs are small non-coding RNAs that participate in different biological processes, providing subtle combinational regulation of cellular pathways, often by regulating components of signalling pathways. Aberrant expression of miRNAs is an important factor in the development and progression of disease. The canonical myomiRs (miR-1, -133 and -206) are central to the development and health of mammalian skeletal and cardiac muscles, but new findings show they have regulatory roles in the development of other mammalian non-muscle tissues, including nerve, brain structures, adipose and some specialised immunological cells. Moreover, the deregulation of myomiR expression is associated with a variety of different cancers, where typically they have tumor suppressor functions, although examples of an oncogenic role illustrate their diverse function in different cell environments. This review examines the involvement of the related myomiRs at the crossroads between cell development/tissue regeneration/tissue inflammation responses, and cancer development.
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BACKGROUND: Exposure to aminoglycoside antibiotics can induce ototoxicity in genetically susceptible individuals carrying certain mitochondrial DNA (mtDNA) mutations (C1494T and A1555G), resulting in hearing loss. So, a rapid diagnostic approach is needed to accurately identify subjects carrying such gene mutations. METHODS: In the present study, we describe a rapid and reliable four-color, real-time quantitative polymerase chain reaction (qPCR) assay for simultaneously detecting two mtDNA 12S rRNA gene variants, A1555G and C1494T, which are prevalent in the Han Chinese population. This multiplex assay incorporates three allele-specific TaqMan probes labeled with different fluorophores in a single reaction, providing high genotyping accuracy for clinical blood samples. RESULTS: Tests with C1494T, A1555G and wild-type DNA exhibited high sensitivity, specificity, reproducibility and accuracy of discriminating mutations from wild-type. CONCLUSIONS: This study shows that this simple and inexpensive method can be used for routine molecular diagnostics and potentially for large-scale genetic screening.
Assuntos
DNA Mitocondrial/genética , Testes Genéticos/métodos , Perda Auditiva Neurossensorial/induzido quimicamente , Reação em Cadeia da Polimerase Multiplex , Mutação , RNA Ribossômico/genética , Reação em Cadeia da Polimerase em Tempo Real , Marcadores Genéticos , Predisposição Genética para Doença , Perda Auditiva Neurossensorial/genética , Humanos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Type 2 diabetes (T2DM) is a complex multifactorial metabolic disorder that affects >100 million individuals worldwide, yet the mechanisms involved in the development and progression of the disease have not yet been fully elucidated. The present study examined the mRNA and micro (mi)RNA expression profiles by microarray analysis in the pancreas islets of spontaneously diabetic Goto-Kakizaki rats with the aim to identify regulatory mechanisms underlying the pathogenesis of T2DM. A total of 9 upregulated and 10 downregulated miRNAs were identified, including miR-150, miR-497, miR-344-3p and let-7f, which were independently validated by quantitative polymerase chain reaction assays. In addition, differential expression of 670 genes was detected by mRNA microarray analysis, including 370 upregulated and 247 downregulated genes. The differentially expressed genes were statistically associated with major cellular pathways, including the immune response pathway and the extracellular matrix (ECM)-receptor interaction pathway. Finally, a reverse regulatory association of differentially expressed miRNAs and their predicted target genes was constructed, supported by analysis of their mRNA and miRNA expression profiles. A number of key pairs of miRNA-mRNA was proposed to have significant roles in the pathogenesis of T2DM rats based on bioinformatics analysis, one example being the let-7f/collagen, type II, alpha 1 pair that may regulate ECM-receptor interactions.
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Perfilação da Expressão Gênica , Ilhotas Pancreáticas/metabolismo , MicroRNAs/genética , RNA Mensageiro/genética , Animais , Diabetes Mellitus Tipo 2/genética , Modelos Animais de Doenças , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Ilhotas Pancreáticas/patologia , Masculino , Ratos , Reprodutibilidade dos TestesRESUMO
BACKGROUND: miR-34a is an important tumor suppressor gene in various cancer types. But little is known about the dysregulation of miR-34a in tongue squamous cell carcinoma (TSCC). In this study, we investigate the expression and potential role of miR-34a in TSCC. METHODS: We evaluated miR-34a expression and its relationship with clinicopathological characters in 75 pairs of TSCC samples, and confirmed the role of miR-34a for predicting lymph node metastases from a further 15 pairs of paraffin-embedded TSCC specimens with stringent clinicopathological recruitment criteria using quantitative reverse transcription polymerase chain reaction (qRT-PCR). The effects of miR-34a on cell proliferation, migration and invasion were examined in TSCC cell lines using Cell Counting Kit-8 assay, wound healing assay and transwell assay, respectively. The effects of miR-34a on the expression of matrix metalloproteinase (MMP) 9 and 14 were detected by luciferase reporter assays and Western blot analysis. The expression of miR-34a, MMP9 and MMP14 were also confirmed in TSCC samples by in situ hybridization and immunohistochemistry. RESULTS: miR-34a expression in tumor tissues from TSCC patients with positive lymph node metastases was significantly lower than that with negative lymph node metastases. Overexpression of miR-34a significantly suppressed migration and invasion in TSCC cells and simultaneously inhibited the expression of MMP9 and MMP14 through targeting the coding region and the 3'untranslated region, respectively. Moreover, miR-34a expression in TSCC was inversely correlated with protein expression of MMP9 and MMP14 in the TSCC samples. CONCLUSIONS: miR-34a plays an important role in lymph node metastases of TSCC through targeting MMP9 and MMP14 and may have potential applications in prognosis prediction and gene therapy for lymph node metastases of TSCC patients.
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Carcinoma de Células Escamosas/genética , Regulação Neoplásica da Expressão Gênica , Metaloproteinase 14 da Matriz/genética , Metaloproteinase 9 da Matriz/genética , MicroRNAs/genética , Neoplasias da Língua/genética , Regiões 3' não Traduzidas , Idoso , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Feminino , Genes Reporter , Humanos , Luciferases/genética , Luciferases/metabolismo , Linfonodos/metabolismo , Linfonodos/patologia , Metástase Linfática , Masculino , Metaloproteinase 14 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Invasividade Neoplásica , Transdução de Sinais , Neoplasias da Língua/metabolismo , Neoplasias da Língua/patologiaRESUMO
Thermotaxis has been demonstrated to be an important criterion for sperm evaluation, yet clinical assessment of thermotaxis capacity is currently lacking. In this article, the on-chip thermotaxis evaluation of human sperm is presented for the first time using an interfacial valve-facilitated microfluidic device. The temperature gradient was established and accurately controlled by an external temperature gradient control system. The temperature gradient responsive sperm population was enriched into one of the branch channels with higher temperature setting and the non-responsive ones were evenly distributed into the two branch channels. We employed air-liquid interfacial valves to ensure stable isolation of the two branches, facilitating convenient manipulation of the entrapped sperm. With this device, thermotactic responses were observed in 5.7%-10.6% of the motile sperm moving through four temperature ranges (34.0-35.3 °C, 35.0-36.3 °C, 36.0-37.3 °C, and 37.0-38.3 °C, respectively). In conclusion, we have developed a new method for high throughput clinical evaluation of sperm thermotaxis and this method may allow other researchers to derive better IVF procedure.
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MicroRNA miR-26a and long noncoding RNA (lncRNA) MEG3 gene have been independently reported to be tumor suppressor genes in various cancers, but neither has been previously associated with tongue squamous cell carcinoma (TSCC). We report here that miR-26a and lncRNA MEG3 gene expression were both strongly reduced in TSCC compared with levels in matched nonmalignant tissues, and combined low expression levels of both miR-26a and MEG3 emerged as an independent prognostic factor for poor clinical outcome in TSCC patients. Assays in the human TSCC cell lines SCC-15 and CAL27 showed that miR-26a targets the DNA methyltransferase 3B transcript and that its inhibition may result in the upregulation of MEG3, providing a plausible link between the observed reduction of miR-26a and MEG3 in TSCC tissue. Furthermore, the overexpression of miR-26a or MEG3 in SCC-15 and CAL27 cells inhibited cell proliferation and cell cycle progression, and promoted cell apoptosis. Considering the poor prognostic outcomes associated with reduced miR-26a and MEG3, our findings imply that these factors likely play important antitumor effects in TSCC pathogenesis. Furthermore, they represent potential prognostic biomarkers for stratification of TSCC patients.
Assuntos
Carcinoma de Células Escamosas/genética , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , RNA Longo não Codificante/genética , Neoplasias da Língua/genética , Apoptose , Western Blotting , Carcinoma de Células Escamosas/mortalidade , Carcinoma de Células Escamosas/patologia , Ciclo Celular , Proliferação de Células , Células Cultivadas , DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , Feminino , Seguimentos , Humanos , Queratinócitos/citologia , Queratinócitos/metabolismo , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Prognóstico , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Taxa de Sobrevida , Neoplasias da Língua/mortalidade , Neoplasias da Língua/patologia , DNA Metiltransferase 3BRESUMO
Molecular diagnostics using microarrays are increasingly being used in clinical diagnosis because of their high throughput, sensitivity, and accuracy. However, standard microarray processing takes several hours and involves manual steps during hybridization, slide clean up, and imaging. Here we describe the development of an integrated platform that automates these individual steps as well as significantly shortens the processing time and improves reproducibility. The platform integrates such key elements as a microfluidic chip, flow control system, temperature control system, imaging system, and automated analysis of clinical results. Bead labeling of microarray signals required a simple imaging system and allowed continuous monitoring of the microarray processing. To demonstrate utility, the automated platform was used to genotype hereditary hearing-loss gene mutations. Compared with conventional microarray processing procedures, the platform increases the efficiency and reproducibility of hybridization, speeding microarray processing through to result analysis. The platform also continuously monitors the microarray signals, which can be used to facilitate optimization of microarray processing conditions. In addition, the modular design of the platform lends itself to development of simultaneous processing of multiple microfluidic chips. We believe the novel features of the platform will benefit its use in clinical settings in which fast, low-complexity molecular genetic testing is required.
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Automação Laboratorial/métodos , Testes Genéticos/métodos , Perda Auditiva/diagnóstico , Perda Auditiva/genética , Análise em Microsséries/métodos , Técnicas de Diagnóstico Molecular/métodos , Mutação , Ensaios de Triagem em Larga Escala/métodos , Humanos , Manejo de Espécimes/métodosRESUMO
Dielectrophoresis (DEP) can be used to noninvasively measure the dielectric state of the cell, and this data can be used to monitor cell health or apoptosis. In this study, we followed events associated with cytosine arabinoside (Ara-C)-induced apoptosis in NB4 cells using DEP analysis. Our data showed that the membrane capacitance of NB4 cells decreases from 9.42 to 7.63 mF/m(2) in the first 2 hours following treatment with Ara-C, and that this decreased capacitance persists for >12 hours. Additionally, cytoplasmic conductivity decreases from 0.217 to 0.190 S/m within 2 hours of Ara-C treatment; this level is maintained for a short period of time before decreasing. We also investigated these events molecularly at the level of gene expression using microarray analysis and showed that the expression of genes related to membrane capacitance and cytoplasmic conductivity change dramatically as early as 2 hours post-Ara-C treatment, and further demonstrated a temporal relationship between the dielectric properties and key events in apoptosis. This study, integrating physical electrical properties of the cell membrane and cytoplasm with those of conductivity-related gene networks, provides new insights into the molecular mechanisms underlying the initiation of apoptosis, establishing a systematic foundation for DEP application in follow-up drug screening and development of medicines for treating leukemia.
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Apoptose , Citarabina/farmacologia , Eletroforese/métodos , Perfilação da Expressão Gênica/métodos , Anexina A5/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/genética , Apoptose/fisiologia , Benzimidazóis/química , Benzimidazóis/metabolismo , Carbocianinas/química , Carbocianinas/metabolismo , Linhagem Celular Tumoral , Análise por Conglomerados , Condutividade Elétrica , Humanos , Leucemia , Microscopia Eletrônica de Varredura , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Here, we describe a novel oligonucleotide array-based transcription factor (TF) interaction assay platform that can directly identify cointeracting TF complexes following binding to their regulatory DNA elements. This platform that combines immuno-coprecipitation technology with our previously reported oligonucleotide array-based TF assay (OATFA), is named targeted immuno-coprecipitation OATFA (TIC-OATFA). We illustrate use of the system to identify interaction partners of STAT1 (signal transducer and activator of transcription proteins 1) in mouse fibroblasts. Several previously known partners of STAT1, as well as new partners, were identified by TIC-OATFA, including the upstream stimulatory factors 1 and 2 (USF1, USF2), nuclear factor of activated T cells, TATA box-binding protein, nuclear factor erythroid-derived 2, nuclear factor-kappa B, and nuclear factor 1. Both USF1 and nuclear factor-kappa B are well known to interact with STAT1, but the other five TFs are previously unreported STAT1 interaction partners. We examined interactions between one new TF, USF2, and STAT1 in detail. USF2 belongs to the group of bHLH-zip transcription factors, which in a number of diseases including cancers, has enhanced activity. In summary, a novel oligonucleotide array-based assay platform was developed and used to study interactions between STAT1 and functional TF binding partners, revealing that USF2 and potentially four other new TFs are partners of STAT1 in an IFN-γ stimulated mouse fibroblast cell line.
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Análise de Sequência com Séries de Oligonucleotídeos/métodos , RNA Mensageiro/metabolismo , Fator de Transcrição STAT1/metabolismo , Fatores de Transcrição/metabolismo , Animais , Fibroblastos/metabolismo , Células HeLa , Humanos , Imunoprecipitação/métodos , Interferon gama/metabolismo , Camundongos , Células NIH 3T3 , Proteômica/métodos , RNA Mensageiro/análise , RNA Mensageiro/genética , Fator de Transcrição STAT1/análise , Fator de Transcrição STAT1/genética , Fatores de Transcrição/análise , Fatores de Transcrição/genética , Fatores Estimuladores Upstream/metabolismoRESUMO
Hepatitis B virus (HBV) encoded X protein (HBx) has been implicated in apoptotic and related pathogenic events during hepatocellular carcinoma. However, the underlying molecular mechanism through which HBx acts is largely unclear. We used tandem affinity purification under mild conditions to gain insight into the HBx interactome in HBV-producing HepG2.2.15 cells and identified 49 proteins by mass spectrometry that are potentially associated with HBx. Two of the key proteins of the caspase-independent apoptosis pathway were newly identified, apoptosis-inducing factor (AIF) and the homologous AMID (AIF-homologue mitochondrion-associated inducer of death). We confirmed the interactions of HBx with AIF and with AMID by reciprocal coimmunoprecipitation experiments, respectively. We observed the expression of HBx-reduced AIF-mediated apoptosis and HBx colocalization with AIF and AMID, principally in the cytoplasm. Furthermore, the elevated cytoplasmic levels of HBx could inhibit mitochondrion-to-nucleus translocation of AIF. Here, we present the first detailed molecular evidence that HBx can repress apoptosis via inhibition of the caspase-independent apoptosis pathway. This inhibition of apoptosis involves the repression of the mitochondrion-to-nucleus translocation of AIF, although tests with AMID were not conclusive. These findings provide important insights into the new mechanism of the apoptosis inhibition by HBV.
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Apoptose , Caspases/metabolismo , Transativadores/fisiologia , Fator de Indução de Apoptose/isolamento & purificação , Fator de Indução de Apoptose/metabolismo , Proteínas Reguladoras de Apoptose/isolamento & purificação , Proteínas Reguladoras de Apoptose/metabolismo , Cromatografia de Afinidade , Células Hep G2 , Interações Hospedeiro-Patógeno , Humanos , Proteínas Mitocondriais/isolamento & purificação , Proteínas Mitocondriais/metabolismo , Ligação Proteica , Mapeamento de Interação de Proteínas , Transporte Proteico/fisiologia , Transativadores/metabolismo , Proteínas Virais Reguladoras e AcessóriasRESUMO
Nephrotoxicity is one of the major concerns for anticancer drug safety because most drugs are metabolized and excreted by the kidneys. Convenient tools able to perform rapid in vitro cytotoxicity analysis and identify drug side effects in kidney cells during early phases of drug discovery could be beneficial to drug development programs. Here we developed an electrical cell-substrate impedance sensing system (ECIS) capable of continuously measuring the dosage and time response of human proximal tubular epithelial (HK2) cells exposed to four drugs throughout the experimental period. These drugs induced HK2 cell apoptosis/death in a dose-dependent manner, although with very different dose-response effects. DDP (50 µM) was the most cytotoxic and induced obvious HK2 cell apoptosis rapidly after exposure. The other three drugs had much lower cytotoxicity, even at concentrations approaching 1 mM. The results obtained from our ECIS system correlated well with conventional in vitro assays such as flow cytometry and cell viability assays. Notably, the continuous and automatic measurements provided by ECIS system allow for better resolution for drugs with different temporal toxicity profiles. Furthermore, we investigated the effect of DDP's antidotes, glutathione and sodium subsulfite, on DDP-induced cytotoxicity, both of which decreased nephrotoxicity of DDP in a dose-dependent manner. Overall this study illustrates the convenience of ECIS for direct, continuous assessment of the cytotoxicity of anticancer drugs in vitro. ECIS has the potential to become a useful, non-invasive analytical method for early evaluation of drugs and antidotes of toxins.
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Antineoplásicos/efeitos adversos , Antineoplásicos/farmacologia , Impedância Elétrica , Células Epiteliais/efeitos dos fármacos , Rim/citologia , Rim/efeitos dos fármacos , Animais , Antídotos/farmacologia , Apoptose/efeitos dos fármacos , Bioensaio/instrumentação , Bioensaio/métodos , Carboplatina/efeitos adversos , Carboplatina/farmacologia , Linhagem Celular/efeitos dos fármacos , Cisplatino/efeitos adversos , Cisplatino/farmacologia , Ciclofosfamida/efeitos adversos , Ciclofosfamida/farmacologia , Relação Dose-Resposta a Droga , Células Epiteliais/citologia , Fluoruracila/efeitos adversos , Fluoruracila/farmacologia , Glutationa/farmacologia , Humanos , Rim/metabolismo , Túbulos Renais/citologia , Tiossulfatos/farmacologiaRESUMO
In this paper, we describe a comprehensive general system adapted for quantitative fluorescence resonance energy transfer (FRET) measurement using signals from three channels of a fluorescence instrument. The general FRET measurement system involves two established methods, as well as two novel approaches. Unlike the previous measurements, which can be taken correctly only when the quantity of the acceptor is greater than or equal to that of the donor, one of our novel methods can overcome this obstacle and take quantitative FRET measurements when the donor is in excess of the acceptor. Hence the general FRET measurement system allowed one to determine the exact distance when the donor and acceptor were present in different quantities, and integrated the methods for quantitative FRET measurements. The uniformity of measured values and utility of each method were validated using molecular standards based on DNA oligonucleotide rulers. We also discussed and validated the use of a novel method for estimating the relative quantities of the donor and acceptor fluorophores when they were not known before an appropriate method of this system can be selected.
Assuntos
Transferência Ressonante de Energia de Fluorescência , Oligodesoxirribonucleotídeos/química , DNA , Corantes Fluorescentes , Oligodesoxirribonucleotídeos/síntese químicaRESUMO
PURPOSE: Recent studies have suggested that microRNA biomarkers could be useful for stratifying lung cancer subtypes, but microRNA signatures varied between different populations. Squamous cell carcinoma (SCC) is one major subtype of lung cancer that urgently needs biomarkers to aid patient management. Here, we undertook the first comprehensive investigation on microRNA in Chinese SCC patients. EXPERIMENTAL DESIGN: MicroRNA expression was measured in cancerous and noncancerous tissue pairs strictly collected from Chinese SCC patients (stages I-III), who had not been treated with chemotherapy or radiotherapy prior to surgery. The molecular targets of proposed microRNA were further examined. RESULTS: We identified a 5-microRNA classifier (hsa-miR-210, hsa-miR-182, hsa-miR-486-5p, hsa-miR-30a, and hsa-miR-140-3p) that could distinguish SCC from normal lung tissues. The classifier had an accuracy of 94.1% in a training cohort (34 patients) and 96.2% in a test cohort (26 patients). We also showed that high expression of hsa-miR-31 was associated with poor survival in these 60 SCC patients by Kaplan-Meier analysis (P = 0.007), by univariate Cox analysis (P = 0.011), and by multivariate Cox analysis (P = 0.011). This association was independently validated in a separate cohort of 88 SCC patients (P = 0.008, 0.011, and 0.003 in Kaplan-Meier analysis, univariate Cox analysis, and multivariate Cox analysis, respectively). We then determined that the tumor suppressor DICER1 is a target of hsa-miR-31. Expression of hsa-miR-31 in a human lung cancer cell line repressed DICER1 activity but not PPP2R2A or LATS2. CONCLUSIONS: Our results identified a new diagnostic microRNA classifier for SCC among Chinese patients and a new prognostic biomarker, hsa-miR-31.
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Carcinoma de Células Escamosas/genética , Neoplasias Pulmonares/genética , MicroRNAs/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma de Células Escamosas/diagnóstico , Carcinoma de Células Escamosas/metabolismo , Linhagem Celular Tumoral , RNA Helicases DEAD-box/biossíntese , RNA Helicases DEAD-box/genética , Feminino , Perfilação da Expressão Gênica , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/metabolismo , Masculino , MicroRNAs/biossíntese , Pessoa de Meia-Idade , Prognóstico , Proteína Fosfatase 2/genética , Proteínas Serina-Treonina Quinases/genética , Ribonuclease III/biossíntese , Ribonuclease III/genética , Taxa de Sobrevida , Transfecção , Proteínas Supressoras de Tumor/genética , Adulto JovemRESUMO
MiRNAs (microRNAs) are small non-coding RNAs involved in mammalian gene expression of cellular processes including differentiation, apoptosis and cancer development. Both specific miRNAs and mRNAs have been identified during monocytic differentiation, but their interactions have not been fully characterized. Here we report that by genome-wide microarray analysis for U937 monocytic differentiation induced by TPA, a large number of miRNAs and mRNAs were differentially expressed, and by bioinformatics analysis could demonstrate that their functional pathway patterns overlap strongly. While expected negative correlation between the expression levels of miRNAs and their target mRNAs was seen, several positive correlations between miRNAs and host mRNAs were also observed, such as C13orf25/miR17, MCM7/miR93, and MGC14376/miR22. These microarray data were verified by quantitative RT-PCR, and the TPA-induced differentiation of U937 cells was confirmed by flow cytometric analysis. Our study suggests an intrinsic correlation between miRNAs and mRNAs underlying their interactions which would provide new insights for defining the mechanisms occurring during monocytic differentiation.
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Diferenciação Celular/genética , Perfilação da Expressão Gênica , MicroRNAs/genética , Monócitos/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , Carcinógenos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica , Humanos , Monócitos/efeitos dos fármacos , Acetato de Tetradecanoilforbol/farmacologia , Células U937RESUMO
UNLABELLED: MicroRNAs (miRNAs) play important roles in the posttranscriptional regulation of gene expression. Recent evidence has indicated the pathological relevance of miRNA dysregulation in hepatitis virus infection; however, the roles of microRNAs in the regulation of hepatitis B virus (HBV) expression are still largely unknown. In this study we identified that miR-373 was up-regulated in HBV-infected liver tissues and that the members of the miRs-371-372-373 (miRs-371-3) gene cluster were also significantly co-up-regulated in HBV-producing HepG2.2.15 cells. A positive in vivo association was identified between hepatic HBV DNA levels and the copy number variation of the miRs-371-3 gene cluster. The enhanced expression of miRs-372/373 stimulated the production of HBV proteins and HBV core-associated DNA in HepG2 cells transfected with 1.3×HBV. Further, nuclear factor I/B (NFIB) was identified to be a direct functional target of miRs-372/373 by in silico algorithms and this was subsequently confirmed by western blotting and luciferase reporter assays. Knockdown of NFIB by small interfering RNA (siRNA) promoted HBV expression, whereas rescue of NFIB attenuated the stimulation in the 1.3×HBV-transfected HepG2 cells. CONCLUSION: Our study revealed that miRNA (miRs-372/373) can promote HBV expression through a pathway involving the transcription factor (NFIB). This novel model provides new insights into the molecular basis in HBV and host interaction.
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Vírus da Hepatite B/fisiologia , MicroRNAs/fisiologia , Fatores de Transcrição NFI/fisiologia , Regiões 3' não Traduzidas/fisiologia , DNA Viral/análise , Dosagem de Genes , Células Hep G2 , Humanos , Fatores de Transcrição NFI/antagonistas & inibidoresRESUMO
Nucleotide analogues are used increasingly in medicine and biotechnology to effect DNA sequence change, principally via clastogenic and transcriptional effects. This article, however, discusses the use of mutagenic nucleotide analogues to improve the sequencing of recalcitrant and repetitive DNA motifs. Guidance in the technical and practical approaches that support use of this approach with different DNA sequencing technologies is provided, including for high-throughput technologies.
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DNA/genética , Análise de Sequência de DNA , Mutação , Reação em Cadeia da PolimeraseRESUMO
Differential gene expression is tightly controlled by transcription factors (TFs), which bind close to target genes and interact together to activate and coregulate transcription. Bioinformatics analysis of published genome-wide gene expression data has allowed the development of comprehensive models of TFs likely to be active in particular tissues (signature TFs); however, the predicted activities of many of the TFs have not been experimentally confirmed. Here, we describe methods for the parallel analysis of the activities of more than 200 transcription factor proteins, using an advanced oligonucleotide array-based transcription factor assay (OATFA) platform, to assay TF activities in mice. The system uses a PCR-based system to translate cellular levels of target DNA-TF complex into a dye-tagged DNA signal, which is read by the developed microarray. The PCR step introduces semiquantitative amplification of the represented TF binding sequences. Experimental OATFA findings can identify many TF activities, which bioinformatics profiling does not predict. Newly identified TF activities can be confirmed by antibody-ELISA against active TFs. The PCR-based OATFA microarray analysis is a comprehensive method that can be used to reveal transcriptional systems and pathways which may function in different mammalian tissues and cells.
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DNA/metabolismo , Reação em Cadeia da Polimerase/métodos , Fatores de Transcrição/metabolismo , Animais , CamundongosRESUMO
In vitro fertilization (IVF) therapy is an important treatment for human infertility. However, the methods for clinical IVF have only changed slightly over decades: culture medium is held in oil-covered drops in Petri dishes and manipulation occurs by manual pipetting. Here we report a novel microwell-structured microfluidic device that integrates single oocyte trapping, fertilization and subsequent embryo culture. A microwell array was used to capture and hold individual oocytes during the flow-through process of oocyte and sperm loading, medium substitution and debris cleaning. Different microwell depths were compared by computational modeling and flow washing experiments for their effectiveness in oocyte trapping and debris removal. Fertilization was achieved in the microfluidic devices with similar fertilization rates to standard oil-covered drops in Petri dishes. Embryos could be cultured to blastocyst stages in our devices with developmental status individually monitored and tracked. The results suggest that the microfluidic device may bring several advantages to IVF practices by simplifying oocyte handling and manipulation, allowing rapid and convenient medium changing, and enabling automated tracking of any single embryo development.
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Embrião de Mamíferos , Fertilização in vitro/instrumentação , Microfluídica/instrumentação , Oócitos , Meios de Cultura , Análise de Elementos Finitos , HumanosRESUMO
The accurate detection of mycobacterial species from isolates and clinical samples is important for pathogenic diagnosis and treatment and for disease control. There is an urgent need for the development of a rapid, simple, and accurate detection method. We established a biochip assay system, including a biochip, sample preparation apparatus, hybridization instrument, chip washing machine, and laser confocal scanner equipped with interpretation software for automatic diagnosis. The biochip simultaneously identified 17 common mycobacterial species by targeting the differences in the 16S rRNA. The system was assessed with 64 reference strains and 296 Mycobacterium tuberculosis and 243 nontuberculous mycobacterial isolates, as well as 138 other bacteria and 195 sputum samples, and then compared to DNA sequencing. The entire biochip assay took 6 h. The concordance rate between the biochip assay and the DNA sequencing results was 100%. In conclusion, the biochip system provides a simple, rapid, reliable, and highly accurate clinical assay for determination of mycobacterial species in a 6-h procedure, from either culture isolates or sputum samples, allowing earlier pathogen-adapted antimicrobial therapy in patients.
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Técnicas Bacteriológicas/métodos , Mycobacterium tuberculosis/classificação , Mycobacterium tuberculosis/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Escarro/microbiologia , Tuberculose/microbiologia , Humanos , RNA Bacteriano/genética , RNA Ribossômico 16S/genética , Fatores de TempoRESUMO
Two-color DNA microarray platforms are widely used for determining differential amounts of target sequences in parallel between sample pairs. However, the fluorescence (or Forster) resonance energy transfer (FRET) between two fluorophores can potentially result in the distortions of the measured fluorescence signals. Here we assessed the influence of FRET on the two-color DNA microarray platform and developed a reliable and convenient method for the correction of FRET distortion. Compared to current methods of normalization based on the statistical analysis and the hypothesis that only a small part of target sequences are differentially presented between sample pairs, our FRET correction method can recover the undistorted signals by the compensation of fluorescence emission, without considering the number of target sequences differentially presented. The correction method was validated with samples at different target ratios and with microarrays spotted in different probe concentrations. We also applied the FRET correction method to gene expression profiling arrays, and the results show that FRET was present when the content of target sequence was beyond a threshold amount and that the process incorporating our FRET correction method can improve the reliability of the gene expression profiling microarray platform in comparison with the current process without FRET correction.